1. Field of the Invention
This invention relates to the field of oligonucleotide delivery systems and gene therapy. In particular, this invention is directed to a self-assembling polynucleotide delivery system comprising a polynucleotide and a dendrimer polycation, and optionally other agents, aiding the delivery of the polynucleotide to a desired subcellular-location. The polynucleotide and the other agents are in general associated with the polynucleotide via non-covalent interactions. Agents suitable for use herein include DNA-masking components, cell recognition agents, charge-neutralization agents, membrane-permeabilization agents, and subcellular localization agents, among others.
2. Description of the Background
Molecular biologists have identified the chromosomal defects in a large number of human hereditary diseases, raising the prospects for cures using gene therapy. This emerging branch of medicine aims to correct genetic defects by transferring cloned and functionally active genes into the afflicted cells. Cystic fibrosis (CF) is a fatal recessive genetic disease characterized by abnormalities in chloride transport. The locus of the disease has been traced to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Correction of the underlying gene defect by complementation or replacement of the defective CFTR is the ultimate cure for CF.
Gene therapy or the in vivo delivery and expression of genes is a fast-developing science that can be used to replace defective genes. Several systems and polymers suitable for the delivery of polynucleotides are known in the art. Among them, viral vectors such as adenoviral vectors have been used to transfer CFTR to the cotton rat lung in vivo. Although high levels of in vivo transfection have been reported with the adenoviral vectors, non-viral delivery systems have a number of advantages for the delivering of polynucleotides.
During the past decade, a number of methods have been developed to introduce functional genes into mammalian cells in vitro. These techniques are applicable to gene therapy, provided that the target cells can be removed from the body, transfected, and the transfected cells amplified and then returned to the patient. This, however, is not possible for CF patients.
At present, the best in vivo transfection efficiencies are obtained with retroviruses. However, the efficiency of transfection is variable and virus based gene delivery systems have the risk of causing viral infection or cancer. Clearly, although no acute complications have been observed stemming from the use of retroviral vectors in humans, the possibility of long-term complications mandate careful patient monitoring.
The potential risks of using viral based vectors and the conceptual advantages of using instead plasmid DNA constructs for gene therapy have led to the development of various physical and chemical methods for aiding gene transfer in the absence of viral vectors. The most intensely studied systems involve the treatment of cells with calcium phosphate or a cationic facilitator. Other methods involve the injection of the DNA during physical puncturing of the membrane or passive uptake of the DNA during permeabilization or abrasion of the cellular membrane. Each of the these methods is intrinsically aggressive and is not preferred for in vivo use.
The use of a direct gene delivery system that does not involve the use of viral vehicles may avoid the risks posed by viral systems. A non-viral carrier suitable for gene delivery must be able to surmount many barriers. It must survive in the circulation and be able to target the cell of choice, to introduce DNA into the cytoplasm of the cell, and to transport the DNA into the nucleus.
At present, viruses are the most efficient vectors for gene transfer, but the potential risks associated with their use have catalyzed a search for synthetic DNA-delivery systems. Early work showed that polycations such as polylysine and DEAE-dextran promote the uptake of proteins and single- and double-stranded polynucleotides into animal cells, and since then, polylysine-based vectors have been extensively tested for gene transfer. However, these polycations are relatively cytotoxic and by themselves not very efficient, which limits their usefulness for transfecting cells in culture.
In spite of these drawbacks, polylysines have a number of advantages such as helping to
1) assemble DNA into a compact structure, PA1 2) destabilize cell membranes, and PA1 3) provide a handle for the attachment of other effectors to the nucleic acid. PA1 1) DNA-masking agents. PA1 2) Cell recognition agents. PA1 3) Charge-neutralization and membrane-permeabilization agents. PA1 4) Subcellular localization agents.
The neutralization and condensation of DNA by polylysines into small (ca 100 nm) toroid-like structures, promotes the endocytosis of the nucleic acid into cells in vitro. The endocytic process may be further stimulated by the covalent attachment to the polycation of specific ligands like transferrin, asialoorosomucoid or insulin. When polycation transfection procedures are based upon receptor-mediated or fluid phase endocytosis, a large fraction of the endocytosed DNA becomes trapped in intracellular vesicles and is ultimately degraded in the lysosomes. Lysosomal degradation can be partially bypassed by the addition of lysosomotrophic agents such as chloroquine during transfection, or by attachment of endosome disrupting agents, such as inactivated viruses or viral fusogenic peptides to the polylysine. The ability of polylysine-DNA complexes to transfect cells is strongly dependent upon the presence of these effectors.
One form of protecting the polynucleotide in the circulation, so that it survives long enough to arrive at the desired cellular destination, is the "masking" or protecting of the polynucleotide.
Microparticulates, such as erythrocyte ghosts, reconstituted viral envelopes and liposomes have been used in part as protection in gene transfer. A successful liposome system uses the cationic lipid N-[1(-2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), mixed with phosphatidylethanolamine (PE) to form the reagent Lipofectin.TM.. Lipofectin.TM. is a cationic liposome which may be mixed with the DNA, and the mixture added to a cell without need for encapsulation of the DNA inside the liposome with cationic reagents. Lipofectin.TM. has been used to transfect reporter genes into human lung epithelial cells in culture, to introduce the chloramphenicol acetyltransferase (CAT) gene into rat cells by the intratracheal route, and to introduce the CAT gene into mice cells by the intratracheal and intravenous routes. In the case of the CAT gene, about 50% of the airway epithelial rat cells transiently expressed the .beta.-galactosidase reporter gene, although the level of expression was not quantitated. When the CAT gene was attached to a steroid sensitive promoter and transfected into rat lung, its expression was shown to be positively regulated by dexamethasone. Cytotoxicity, however, is a definite problem when high concentrations of Lipofectin.TM. are used.
Substitutes for DOTMA including lipopolyamine, lipophilic polylysines, and a cationic cholesterol have been used to mediate gene transfer in culture. Although some of these show an about three fold improvement over the transfection rates observed with Lipofectin.TM., their toxicity remains a problem. The mechanism responsible for transfection using cationic lipids has not been thoroughly explored. The past approach has been to synthesize different cationic lipids and try them in transfection assays, rather than to systematically study how the delivery systems introduce DNA into a cell. The finding that DOTMA/PE liposomes can undergo bilayer fusion with anionic liposomes suggests that DOTMA may facilitate the direct entry of the DNA via the plasma membrane. On the other hand, for high efficiency transfection cationic lipids systems require PE, possibly because PE can form intramembrane lipid intermediates which facilitate membrane fusion.
Efficient gene transfer also requires the targeting of the DNA to the cell of choice. Various procedures based upon receptor mediated endocytosis have recently been described for gene transfer. A cell-specific ligand-polylysine conjugate was bound to nucleic acids through charge interactions, and the resulting complex was taken up efficiently by the target cells, such as in the case of the human hepatoma cell line HepG2 and of rat hepatocytes in vivo using this delivery system with asialoorosomucoid as a ligand. The stable expression of an enzymatic activity in HepG2 cells following insulin-directed targeting as well as the transferrin-polycation-mediated delivery of a plasmid into the human leukemic cell line K-562 and the subsequent expression of the encoded luciferase gene, have been reported. However, the described delivery systems require the linking of high molecular weight targeting proteins to DNA through a polylysine linker. These large ligand-polycation conjugates are heterogenous in size and composition, chemically ill-defined, and difficult to prepare in a reproducible fashion. Moreover, in many of the receptor mediated systems, chloroquine or other disruptors of intracellular trafficking are required for high levels of transfection. In one study, an adenovirus was used to enhance gene delivery of a receptor mediated system.
Thus, genes can be delivered into the interior of mammalian cells by receptor mediated endocytosis, with a fraction of the exogenous DNA escaping degradation, entering the nucleus, and being expressed. The level of expression, however, is low, probably due to the limited entry of the foreign DNA into the cytoplasm.
The direct delivery of genes is also aided by neutralization of the large negative charge on the polynucleotide, and the (often concomitant) ability to permeabilize the membrane of the targeted cell. The use of polycations to neutralize the polynucleotide charge aids the permeabilization of the membrane and the translocation of the polynucleotide. Cationic lipids have also been used for this purpose. Certain cationic lipids termed lipopolyamines and lipointercalants are also known.
Once the polynucleotide has entered the targeted cell, the direct delivery of genes may be aided by directing the genes to the proper subcellular location. One obvious target for the delivery of deoxyribonucleotides is the nucleus. Ligands known to aid in this process are nuclear localization peptides or proteins containing nuclear localization sequences.
The transfection efficiency obtained with reconstituted viral envelopes was shown to increase when the foreign gene is co-delivered into the target cells with nuclear proteins. DNA mixed with nuclear proteins was shown to exhibit a modest increase in transfection over DNA mixed with albumin used as control. Thus, the DNA appears to be incorporated into the nucleus more readily when proteins containing the nuclear localization sequence (NLS) pro-lys-lys-lys-arg-lys-val is associated with the plasmid since the presence of the NLS on a protein designates it for transport through the nuclear pore. Nuclear localization sequences of 14 amino acids have been attached to a variety of macromolecules and even to gold particles (150 .ANG. diameter) and, when introduced into the cytoplasm, they were rapidly incorporated into the nucleus. Nuclear entry appears to be the rate limiting step for successful, stable transfection. This is supported by the finding that when plasmid DNA is microinjected into the cytoplasm, it is unable to bring about cell transfection. No transfection occurred out of 1000 cytoplasmic injections, whereas the microinjection of plasmids directly into the nucleus results in transfection in greater than 50% of the microinjected cells.
The transfection efficiency was also shown to increase when the DNA is condensed using various cationic proteins. Although the reason why DNA condensation increases transfection is not readily apparent, it may be due to an increase in the cellular uptake of DNA or to a decrease in the susceptibility of the DNA to nucleases, which may result in higher amounts of intact DNA in the cell.
The direct delivery of genes associated with one of the above-discussed classes of agents, is further aided by the ability of those agents to remain associated with the DNA. Examples of this are the association of a receptor ligand with a polynucleotide by covalent attachment of the ligand to the polycation polylysine, and optionally by covalently attaching the ligand to a DNA intercalator, e.g., ethidium homodimer (5,5'-diazadecamethylenebis (3,8-diamino-6-phenylphenanthridium) dichloride dihydrochloride) and the association of photoaffinity labels to DNA by covalent attachment to 9-aminoacridine and certain bis-acridines.
Dendrimers are bulky three-dimensional polymers built by reiterative reaction sequences around a core molecule that may be prepared in varied molecular weights and sizes (Tomalia, D. A., et al., "Starburst Cascade Polymers: Molecular-Level Control of Size, Shape, Surface Chemistry, Topology, and Flexibility from Atoms to Macroscopic Matter", Angew. Chem. Int. Ed. Engl. 29:138 (1990)).
However, in the quest for attaining better results in the field of gene therapy, there is still a need for improved polynucleotide delivery systems of high transfection efficiency without the drawbacks of prior systems.